Project Summary Gram-negative bacteria, such as E. coli, have an outer membrane which provides nutrients and added protection for the cell. The outer membrane also contains proteins that can enable pathogenic bacteria to evade host immune responses and cause a number of serious infectious diseases. Despite the importance of these outer membrane proteins (OMPs) for cell survival and virulence, little is known about how these proteins are folded and inserted into the outer membrane. However, in E. coli, we know that a 200 kDa five-component complex called the ?-barrel assembly machinery (BAM) complex mediates the biogenesis of these membrane proteins. In the last decade, the individual structures of components of the BAM complex have been reported with the structure of BamA, the central component, providing the most useful clues to how the BAM complex may function. Here, it was revealed that BamA may destabilize the local membrane, thereby promoting protein folding and insertion into the membrane. Recently, our work and others? have determined the structure of the BAM complex, revealing unprecedented conformational changes within the barrel domain of BamA. Our work here will utilize these structures to study the role of the conformational changes for the function of the BAM complex in OMP biogenesis. Additionally, we will determine how substrate OMPs are recognized by the BAM complex and how they interact with BAM during folding into the membrane. Lastly, we will use structural biology methods to study the BAM complex in a lipid bilayer and in the presence of substrates. Our proposed studies will provide crucial functional and structural insight towards unraveling how the BAM complex performs its essential role in bacteria.